Browsing by Subject "Biomedical Sciences"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Identification of Novel Salmonella Typhimurium Genes Required For Survival During Intestinal Inflammation(May 2013) Endicott-Yazdani, TianaBovine ligated ileal loops provide the best model to examine Salmonella Typhimurium genes required for survival during the early stages of infection, as humans and cattle develop very similar intestinal pathogenesis in response to the organism. Utilizing pools of mutants, both single gene and multi-gene deletion mutants, much of the S. Typhimurium genome can be screened simultaneously. A library consisting of multi-gene deletion mutants was screened in ligated ileal loops in calves. Regions of the genome required for survival in this model were identified in mucus and tissue samples using microarray analysis. STM1187-90 was one such region identified as under selection in both mucus and tissue. A ASTM1188 mutant was confirmed to poorly colonize the intestinal mucus and tissue in the presence of inflammation in competitive infections with the wild type. Complementation in trans reversed the phenotype of the ASTM1188 mutant. The phenotype of the ASTM1188 mutant was replicated and complemented in trans in the murine colitis model. STM1188 is a Salmonella specific gene whose protein product we show to be located in the inner membrane. This gene is absent in host-adapted serovars S. Typhi and S. Paratyphi A. Mutation of the putative lipobox cysteine to alanine resulted in mis- localization of STM1188C24A to the cytoplasm, and the inability to complement ASTM1188 in trans in mice. A second screen of a single gene deletion pool (SGD) identified many novel genes with potential roles during inflammation as well as many predicted genes with defined roles during the early stages of infection. Several novel gene phenotypes were confirmed and subsequently complemented in competitive infections with wild type. AhilE was identified during SGD screening and confirmed in bovine ligated ileal loops as being selected against during competitive infections with extensive inflammation, however, it was not selected against when the inflammatory immune response was limited. HilE has been previously shown to negatively regulate SPI-1 expression. Utilizing the murine colitis model, the AhilE phenotype was confirmed and complemented during competitive infection with wild type. By using (3-galactosidase assays, AhilE was confirmed to overexpress SPI-1, however, surprisingly AhilE also overexpressed SPI-2 during SPI-1 inducing conditions. In the current studies we performed Salmonella screens in bovine ligated ileal loops and confirmed novel virulence genes required for survival during inflammation.Item Nicotine Stimulates Biliary Proliferation and Fibrogenesis and Potentiates Cholangiocarcinoma Growth(May 2013) Jensen, Kendal JayNicotine is a major addictive substance in cigarette smoke, and has powerful biological properties throughout the body. Studies have demonstrated that nicotine promotes the progression of a number of disease processes, including cancer and fibrosis. Recent evidence suggests that nicotine plays a role in liver fibrosis. Cholangiocytes are the epithelial cells that line the intrahepatic and extrahepatic bile ducts. During biliary injury that occurs with diseases such as primary sclerosing cholangitis (PSC), cholangiocytes undergo a reactive process termed "ductular reaction," and display a neuroendocrine phenotype that may contribute to the fibrotic pathologies, and eventual development of cholangiocarcinoma. Epidemiological data indicate that smoking contributes to biliary fibrosis and cholangiocarcinoma. Due to this reason and the prevalence of nicotine usage, it was important to understand the role that nicotine plays in the progression of biliary fibrosis, and the regulators of this process. We first examined whether nicotine has pro-proliferative effects on the biliary system. We demonstrated in vitro that nicotine increases the proliferation of the normal cholangiocyte cell line (NRIC) by MTS assay and PCNA expression by western blot. We also demonstrated that nicotine works through a calcium/inositol triphosphate/phosphorylated extracellular regulated kinase (Ca2+/IP3/p-ERK) intracellular signaling pathway in NRIC. In vivo, rats were treated with nicotine via osmotic minipump infusion for two weeks. In the nicotine-treated group, we found an increase in the proliferation of cholangiocytes identified by cytokeratin (CK-19, a cholangiocyte specific marker) staining, and proliferating cell nuclear antigen (PCNA) expression. Increased expression of fibrotic genes/proteins was seen in liver tissue, and cholangiocytes isolated from rodent livers. Next, we sought to determine the effect of nicotine on cholangiocarcinoma proliferation. In vitro, nicotine stimulated the proliferation of cholangiocarcinoma growth, in the MzChA-1, CCLP1, and HuCC-Tl cell lines. In a nude mouse xenograft model, nicotine treatment caused an increase in tumor volume and PCNA expression. These data support the current literature suggesting that nicotine may induce fibrogenesis, and contributes to the liver pathology observed in smokers. They also support the notion that nicotine stimulates the growth of biliary cancer and call for further investigation of the subject.Item The Role of Specificity Protein Transcription Factors And Noncoding Rnas In Colorectal And Hepatocellular Carcinomas(May 2013) Gandhy, ShrutiSpecificity protein transcription factors, Spl, Sp3 and Sp4 are overexpressed in multiple cancer cell lines and tumor types and Sp-regulated genes play an important role in cancer cell proliferation, survival, angiogenesis and inflammation. Noncoding RNAs have been implicated in carcinogenesis and cancer progression, and our studies investigated the role of Sp proteins and noncoding RNAs in colorectal and hepatocellular carcinomas. We investigated the anticancer activity of curcumin and several synthetic analogs in colon cancer cells. Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates Spl, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. The IC50 (half- maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 /zM for curcumin to 0.7 //M for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Spl, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFkB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Spl, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR- 17-5p that regulate these repressors. These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR- ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes. In our second study, we investigated the effects of Sp proteins and Sp-regulated long noncoding RNAs (IncRNAs) in hepatocellular carcinoma (HCC). LncRNAs have recently garnered attention because of their pivotal role in cancer and other diseases, and IncRNA HULC (Highly Upregulated in Liver Cancer) was identified as a liver cancer- specific noncoding RNA and the most upregulated gene in HCC. Both HULC and Spl are upregulated in HCC, but the relationship between HULC and Spl and the underlying mechanism by which HULC contributes to HCC metastasis have not been investigated. We examined the expression of Sp proteins and IncRNAs in three HCC cell lines and found that HULC is an Spl-regulated IncRNA involved in HCC growth and metastasis. Both Spl and HULC contribute to epithelial to mesenchymal transition (EMT) of HCC cells and downregulation of either resulted in reversal of EMT and acquisition of epithelial characteristics. Our findings identify a novel role for HULC and, as an Spl- regulated IncRNA, make it an attractive target for drug treatment in HCC. Together, these studies provide conclusive evidence regarding the pro-oncogenic role of Sp proteins and Sp-linked ncRNAs, and illustrate the utility of drugs that downregulate Sp proteins as anticancer agents.